Connective tissue substitutes, method of preparation and uses thereof

ABSTRACT

The present invention relates to connective tissue substitute implant and method of preparation thereof. The implant is essentially composed of two bone anchors joined at the proximal ends by matrix layers and/or filaments coated by supplementary biocompatible matrix coating layer which can contain living stem cells isolated from injured connective tissue.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention relates to the field of tissue engineering,production of connective tissue linked to natural bones or syntheticbone substitutes (tendons, ligaments, cartilage, etc.) can benefit fromthe invented procedure. The procedure of the present invention iscarried out to produce bioengineered connective tissue substitutes.Connective tissue substitutes (CTS) of the invention may be constructedfor replacement of ligaments, and most particularly cruciate ligamentsor tendons.

[0003] 2. (b) Description of Prior Art

[0004] Researchers in the surgical arts have been working for many yearsto develop new techniques and materials for use as grafts to replace orrepair damaged or torn tissue structures, particularly bones andconnective tissues, such as ligaments and tendons, and to hasten softtissue repair. It is very common today, for instance, for an orthopedicsurgeon to harvest a central portion of patellar tendon of autogenous orallogenous origin for use as a replacement for a torn cruciate ligament.The surgical methods for such approaches are well known. Further it hasbecome common for surgeons to use implantable prostheses formed fromplastic, metal and/or ceramic material for reconstruction or replacementof physiological structures. Yet despite their wide use, surgicallyimplanted prostheses present many attendant risks to the patient. Itwill suffice to say that surgeons are in need of a non-immunogenic, hightensile strength graft material which can be used for surgical repair ofbones, tendons, ligaments and other functional tissue structures.

[0005] One of the most widely used anterior cruciate ligament (ACL)substitutes is the bone-patellar tendon-bone graft. The centralone-third of the patient's or a donor's patella tendon, along withportions of the bony insertions of the patella tendon, is used as areplacement for the damaged ACL. The bony insertions are harvested asbone fragments to facilitate implantation and fixation of thereplacement graft into osseous tunnels performed in the tibia and femurin the patient's knee joint. The bone-patellar tendon-bone graft is apopular choice for ACL reconstructive surgery because of its high loadstrength after six weeks and its functional bone fixation.

[0006] Some fixation devices employ various structures for coupling witha ligament or a suture and for engaging with the bone. For example, U.S.Pat. No. 5,356,435 discloses an element for fixing a ligament in a bonytunnel. The element includes an internal conduit for receiving an end ofa ligament, and a clamping structure for securing the ligament endwithin the conduit. U.S. Pat. No. 5,356,413 to Martins et al. disclosesa surgical anchor having a body portion and a suture-receiving bone.Another commonly used ACL substitute is the iliotibial band graft. Theiliotibial band is a section of ligament which is harvested from aportion of a patients or a donor's iliotibial ligament located withinthe anterolateral ligament structures of the knee joint. The majorproblem with these techniques is that another part of the body, or thejoint of the donor is often significantly weakened after biopsied to getgrafts. Long term drawbacks of this approach are that chronic pain,patellar fractures, knee instability and cartilage degeneration.

[0007] Researchers have been attempting to develop satisfactory polymersor plastic materials to serve as ligament or tendon for other connectivetissues replacements. It has been found that it is difficult to providelong-term solution using these materials to permanently replaceconnective tissues.

[0008] Artificial materials based on network fibers made of polyester orpolytetrafluoroethylene have been used extensively as replacements forligament and tendon, with some success. However, persistent inflammatoryreactions occur following wear off of particles upon timepost-implantation. Additionally, they do not readily breakdown and arenot readily integrated with the body via remodeling by tissue cells.

[0009] Bioengineered tissues can be used as grafts implants orprostheses to replace damaged tissues.

[0010] U.S. Pat. No. 5,855,619 of Caplan discloses the use of a filamentas load-bearing member of a contracted gel matrix containing mesenchymalcells. The implant described in this patent allows partial repair ofconnective tissues by attaching the implant to the tissue to berepaired. However, since this implant is constructed without anchoringextremities, the anchorage capability is limited.

[0011] Fibroblast-populated Collagen gels (FPCG) constitute aninteresting in vitro model of soft tissues to investigate tissueresponse to various biological, chemical, electrical, and mechanicalstimuli. In the past year, the potential of using a ligament-shaped FPCGto produce a bioengineered anterior cruciate ligament (ACL) has beeninvestigated. Mechanical properties of FPCG are known, however, to besignificantly lower than those required for a functional ACL. Findingways to improve their mechanical properties would be highly beneficialnot only for improving a ACL but also for the tissue engineering fieldin general.

[0012] It is therefore an object of the present invention to provide animplant and method of preparation thereof which obviates thedisadvantages of the prior art approaches.

SUMMARY OF THE INVENTION

[0013] One object of the present invention is to provide an implant forconnective tissue substitution in a human or animal, comprising a pairof bone anchors joined at their proximal ends by at least one supportfilament, the filament being coated by at least one matrix layer ofthickness sufficient to allow for colonization by cells.

[0014] Another object of the present invention is to provide method ofpreparing the implant for connective tissue substitution in an animal,which comprises the steps of providing a set of bone anchors by joininga pair of bone plugs at their proximal ends by at least one supportfilament; and incubating at least one time the set of bone anchors in asolution containing matrix forming molecules for a period timesufficient for the formation of at least one matrix layer around thefilament, the matrix layer with thickness sufficient to allow forcolonization by cells, wherein the incubation is performed undercondition inducing waves, vibration, cyclic traction, and/or statictraction of the implant.

[0015] According to another object of the present invention, there isprovided a method of preparing an implant for connective tissuesubstitution in an animal, said method comprising the steps of:

[0016] a) providing a set of bone anchors by joining a pair of boneplugs at their proximal ends by at least one support filament; and

[0017] b) incubating at least one time the set of bone anchors of stepa) in a solution containing matrix forming molecules for a period timesufficient for the formation of at least one the matrix layer has athickness sufficient to allow for colonization by cells, and wherein theincubation is performed under conditions in which are induced waves,vibrations, cyclic tractions, and/or static tractions of the implant.

[0018] In accordance with the present invention there is provided amatrix which is further colonized by cells. The cells may be autologous,heterologous, or cells selected from the group of fibroblast, myoblast,osteoblast, mesenchymal, endothelial, immune, chondrocyte cell, andcombinations thereof.

[0019] Another object of the invention is to provide with connectivetissue substitution that is partial or complete substitution of aconnective tissue. The connective tissue may be selected from the groupconsisting of tendon, cartilage, disk, meniscus, muscle, tooth, hair,joint, ligament, and combinations thereof

[0020] Furthermore, the filament and/or matrix layer may be dehydratedor lyophilized prior to implantation.

[0021] Also in accordance with the invention, the bone anchor may beselected from the group consisting of bone portion, and piece composedof natural and/or synthetic biocompatible porous material.

[0022] The matrix layer of the invention may be composed of productsselected from the group consisting of chitosan, glycosaminoglycan,chitin, ubiquitin, elastin, polyethylen glycol, polyethylen oxide,vimentin, fibronectin, derivatives thereof, and combination thereof.

[0023] Also, the filament of the present invention may be selected fromthe group of resorbable thread, natural fibers, and filament composed ofproteins, lipids, biocompatible molecules and/or synthetic components.

[0024] The implant of the invention may further comprises apharmaceutically effective amount of biologically active moleculeselected from the group of drugs, growth factors, cytokines,antibiotics, hormones, and combination thereof.

[0025] Another object of the invention is to provide a matrix layerfurther comprising at least one inner layer of gel and/or filamentcoated by at least one supplementary matrix coating layer, or an implantcomprising an inner layer of matrix and/or filament which may bedehydrated or lyophilized prior coating with the supplementary matrixcoating layer. In addition, the matrix-coating layer may furthercomprises cells.

[0026] For the purpose of the present invention the following terms aredefined below.

[0027] The term “matrix” as used herein is intended to mean a network ofbiological extracellular constituents, as example but without limitationcollagen, elastin, fibronectin, laminine, proteoglycans,glycosaminoglycans, chitosan, ubiquitin and derivatives thereof, in ahydrated or dehydrated form. This matrix can be produced with naturalfibers in combination or not with synthetic or semi-synthetic fibers.

[0028] The term “graft”, as used herein refers to a natural and/orsynthetic implantable substitute for various tissue types.

[0029] The term “lyophylization” as used herein is intended to meanpassive or active dehydration of hydrated matrix network as definedabove. Simple air-drying, dessication, vacuum assisted dehydration,warming, water sublimation or other methods may perform thelyophylization.

[0030] The term “chemically fixed” as used herein is intended to meanfixation of treatment of matrix with a chemical, as for example butwithout limiting the invention, paraformaldehyde, ethanol, formaldehyde,methanol, to create link between the matrix fibers and the anchors,bones or bone substitutes.

[0031] This summary of the invention does not necessarily describe allnecessary features of the invention, but that the invention may alsoreside in a sub-combination of these described features. The summary ofthe invention, thus incorporated, presents, therefore, only an examplebut not a limitation of subject matter to exactly this combination offeatures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 illustrates ligament fibroblasts (LF) isolated from a humanACL biopsy;

[0033]FIG. 2 illustrates a transverse hole made in a human bone anchor;

[0034]FIG. 3 illustrates two bone anchors liked with a surgical threadpassed through their transverse holes and twisted;

[0035]FIG. 4 illustrates two sterile bone anchors readily linked bysurgical thread, transferred in a sterile plastic tube and kept inposition by passing a hot metal pin through their transverses holes andacross tube;

[0036]FIG. 5 illustrates an ACL substitute after 24 hours in culture;

[0037]FIG. 6 illustrates an acellular ACL substitute after 24 hours inculture:

[0038]FIG. 7 illustrates an ACL substitute lyophilized;

[0039]FIG. 8 illustrates a histological section of a goat's ACLsubstitute before implantation. The hydrated collagen layer seeded withliving LF surrounds the central circular lyophilized core;

[0040]FIGS. 9A to 9C illustrate the collagen layer of an acellular ACLsubstitute consisting in a network of collagen fibers (A); the adhesionand migration of containing LF into the outer acellular hydratedcollagen layer after 24 hours in culture (B); and same as in b) butafter 48 hours of culture (C);

[0041]FIG. 10 illustrates the multistep procedure to prepare asubstitute ligament; and

[0042]FIG. 11 illustrates an alternative multistep procedure to preparea substitute ligament;

[0043]FIG. 12 and illustrates a macroscopic aspect of a bioengineeredACL ready for implantation (opened goat's knee joint);

[0044]FIG. 13 illustrates a macroscopic aspect of a bioengineered ACLimmediately after implantation in situ (opened goat's knee joint);

[0045]FIG. 14 illustrates the macroscopic aspect of a dehydratedligament substitute;

[0046]FIG. 15 illustrates an histological section of a dehydratedligament substitute showing an alignment of the collagen fibers in itsscaffold (longitudinal plan);

[0047]FIG. 16 illustrates an histological section of an acellularligament substitute grafted in a goat's knee joint for 5 months; and

[0048]FIG. 17 illustrates the macroscopic aspect of a bioengineeredperiodontal ligament ready for implantation.

DETAILED DESCRIPTION OF THE INVENTION

[0049] The following description is of a preferred embodiment by way ofexample only and without limitation to the combination of featuresnecessary for carrying the invention into effect.

[0050] In accordance with one embodiment of the invention, there isprovided an implant allowing permanent implantation of a connectivetissue substitute.

[0051] It is known in the art that synthetic prosthesis such as Dacron™or lad are susceptible indirectly to wear off particles in the kneejoint within a few years, leading to inflammatory reactions, cartilagedegeneration and functional instability of the knee.

[0052] In one embodiment, the implant of the invention doesn't presentthe risks of graft rejection as it is intended to use or integrateautologous cells from the host's connective tissues, their own bonefragments and collagen.

[0053] Another important embodiment of the invention is that the use ofthe instant implant avoids taking any portion of healthy autologoustissues, such as a part of the patellar, semitendinous or TFL iliotibialband, or semimembranous tendons for connective tissue replacement, whichoften lead to chronic pain, muscular weakness or instability of thejoints. Only some cells are removed from its host, defatted if necessaryand processed in one of several well-known procedures used to preparethe tissue for implantation into a human, an animal, as for example butwithout limitation, horses, dogs and other domestic animals. Theinvention applies also in a general manner in the fields ofveterinarian, dentistry, and orthodontic cares.

[0054] The cells useful to contract the collagen fibrils during theformation of an organized tissue-substitute implant can be obtained fromvarious mammalian sources (e.g., bovine, porcine, human, canine). Theconnective tissue cells used in the method of the present invention werefibroblasts, but other mesenchymal cell types, such as fibroblasts ofother sources and tissues may also be used. The human fibroblasts can beisolated by enzymatic disaggregation, explants or perfusion of thetissues of origin.

[0055] Naturally occurring cells in accordance with the presentinvention may include, but not limited to epithelial cells, myoblasts,chondroblasts, osteoblasts, fibroblasts, and other fibrous connectivecells coming from tendon, ligament, cartilage, and the like.

[0056] Also, the autologous connective tissue cells may be conserved ina cell depository to prepare another bioengineered connective tissueimplant for the patients who would break the graft under subsequenttraumatic circumstances.

[0057] In accordance with another embodiment of the present invention,the procedure of implantation may be performed by arthroscopy, avoidingarthrotomies and associated risks (infection, knee pain, and loss ofarticular mobility, major swelling and permanent scar). These advantagescontribute to reduce the cost of medical care on a long-term basis andimprove life quality of the patients post-surgery.

[0058] In another embodiment, a fully functional replacement tissue iswithstand at least the stresses and strains imposed by normal bodilyactivity on the type of tissue the construct is to replace.

[0059] Furthermore, in accordance with one embodiment of the invention,the implant is fully biocompatible and integrable, in vivo, i.e., theimplant resembles a natural tissue so as to be colonized by cells andinteract with these specific cells already present in the body. Thecolonizing cells further organize the implant and secrete specificproducts, such as extracellular matrix constituents, proteins and/orgrowth factors, within the connective tissue substitute of the presentinvention, enabling it to degrade, remodel and regenerate thehistological structures as a functional tissue substitute. Suchintegration may strengthen and conditions the implant to better performsas a substitute tissue.

[0060] Yet in accordance with another aspect of the present invention,the gel layer of the implant may be supplemented with proteins,peptides, or hormones playing roles during tissue integration and tissuerepair. Several known factors may be released from the implant priorimplantation, as, but not limited to growth factors, growth hormones,fibroblast growth factor, epithelial growth factor, TGF-beta, insulin,and IGF-1. Cytokines may be expressed by cells genetically modified,transfected or transformed, to modulate local inflammatory processes,cartilage regeneration vascularisation, etc.

[0061] The collagen can be extracted from various collagen-containinganimal tissues. Examples of possible collagen-containing tissue aretendon, skin, cornea, bone, cartilage, in vertebral disc, cardiovascularsystem and placenta. The collagen used herein is type I collagen. Othertypes of collagen (e.g., type II, III and others) may also be employed.

[0062] In accordance with the most preferred embodiment of the presentinvention, the matrix layer of the implant is composed of Type Icollagen, but can be formed, and is not limited to recombinant collagenproteins as chitosan, chitin, ubiquitin, elastin, polyethylene oxide,vimentin, fibronectin, and combinations thereof.

[0063] According to another aspect of the invention, there is provide animplant having a pair of generally cylindrical bone plug portions joinedat their proximal ends by a core filament, the bone plug preferablyincluding both bone regions.

[0064] In another embodiment of the present invention there is toprovided such an implant in which one of bone anchors is adapted to bepulled through a tunnel in, for example, the femur to allow fusionthereto and the other bone anchor portion is adapted to be pulledthrough a tunnel in the tibia to allow fusion thereto to provide asubstitute for the natural cruciate ligament, the segment being adaptedto be placed under tension between the tunnels to provide a ligamentfunction. Similar procedures may be employed to provide connectivetissue function to other bone joints.

[0065] One other embodiment of this invention is to provide a implantfor promoting the healing and/or regrowth of diseased or damaged tissuestructures by surgically repairing such structures with the implant ofthe invention. The implanted graft is trophic toward vascularization andtissue and may be essentially remodeled to assume the structural andfunctional characteristics of the repaired structure.

[0066] In accordance to another preferred embodiment of the invention,the implant may be lyophilized after its preparation. This processavoids the use of chemicals to strengthen the matrix layer of theimplant, to allow the reinforcement of the links between the bone plugsand the collagen layer polymerized into their trabecular structure.Also, lyophylization permits the preparation of implants addingsuperposed matrix layers to reinforce the structure of a bioengineeredconnective tissue, or conferring a higher resistance to rupture beforeand during surgical implantation procedures.

[0067] Another important embodiment of the invention is thatlyophylization may allow to form matrix layers onto the implant withother biomaterials, as for example, but not limited to elastin, incombination or not with collagen, and replacing the bone anchors of theimplant by other porous anchors, as for example, but not limited tocement, or ceramic.

[0068] It is another object of the present invention to provide a graftimplant which has improved graft fixation capabilities and promotesconnective tissue and bone ingrowth between the graft and the bonytunnel.

[0069] In accordance with the present invention, there is provided adevice and method for cyclic matrix stretching and mechanical testing. Acyclic traction machine is disclosed. In a preferred embodiment, thematrix is maintained in place in the cycling chamber by inserting thetwo bone anchors in metal pins, one fixed to a load cell and the other,attached to a motion controlled cursor. By controlling the position ofthe cursor, the matrix is subjected to cyclic traction with stretchingamplitudes from 0 to 30 mm at a frequency of up to 1 Hz for loweramplitudes, for any extended period of time. The whole system iscontrolled via a LABview VI software. The operator may change easily thetraction conditions and supervise the ongoing tests to make sure thateverything is running smoothly. A set of matrix may be maintained understatic tension, or subjected to a cyclic tension. The cells in a matrixas described in the present invention, may be induced to take astructural organization when submitted to tension stimulus. The stimulusmay be also simply waves in a culture medium by agitation of the petridishes in which is kept a matrix, or an electric stimulus.

[0070] The present invention will be more readily understood byreferring to the following examples which are given to illustrate theinvention rather than to limit its scope.

EXAMPLE I Preparation of Anterior Crutiate Ligament

[0071] Material and Methods

[0072] LF Isolation and Culture

[0073] Torn ACL biopsies are collected from the host. The biopsies arekept at 4° C. for no longer than 24-48 hrs before cell isolation. TheACL biopsy is weighted and cut into small pieces after removal of theperiligamentous tissues. The fragments are digested with 0.125%collagenase containing 2 mM CaCl₂ (1 ml of enzymatic solution/mg oftissue) for 20 hrs, under gentle agitation, at 37° C. A 0.1% trypsinsolution (1 ml/mg of hydrated tissue) is then added to the cellularsuspension for 1 hr. The enzymes are dissolved in Dulbecco'sModification of Eagle's™ medium (Gibco), pH 7.4, containing antibiotics.

[0074] The ligament fibroblasts (LF) isolated from ACL biopsies arecultured in DME supplemented with 10% fetal calf serum (FCS), 100 IU/mlpenicillin G and 25 μg/ml gentamicin (FIG. 1).

[0075] When LF primary cultures reach about 85% confluence, the cellsare detached from their culture flasks using 0.05% trypsin-0.01% EDTAsolution (pH 7.8), for about 10 min at 37° C. The LF suspensions arecentrifuged twice at 200×g for 10 min. The cell pellets are resuspendedin complete culture medium and the cells are counted. The cellularviability is determined using the trypan blue exclusion method

[0076] Up until now, LF were isolated and cultured from ACL biopsies ofmore than 20 patents and 10 animals (goats, dogs, and rabbits) with 100%success. The cells maintained their morphology for more than 7 passagesin culture. For ACL substitutes production, LF cultures from passages 2to 5 are used. Immunofluorescent labeling analysis revealed thatdifferent populations of human LF extracted from ACL biopsies expressvimentin, fibronectin, Types I and III collagens and elastin.

[0077] Preparation of the ACL Substitutes' Bone Anchors

[0078] Bone pieces are washed with ethanol 100% and cut in a cylindricalshape according to dimensions adapted to the needs of the host (averagesize of 1 cm-diam. and 2 cm-long).

[0079] A transverse hole (⅛-in. diam.) is made in each bone anchor (FIG.2). The bone plugs are kept in 100% ethanol overnight to be sterilized.A surgical thread resorbable within 1 month post-surgery, is passedthrough the transverse holes of 2 bone anchors and fixed between thebones by simple stitching. Then the thread is twisted between the bonesto thicken the link (FIG. 3).

[0080] A longitudinal hole or more (1 mm diam. or wider) is made in eachbone anchor. Such holes are drilled in order to increase hydratedcollagen adhesion with the bones. This step is optional. The 2 sterilebone plugs readily linked by the twisted surgical thread are transferredin a sterile plastic tube and kept in position by passing a hot metalpin through their transverse holes and across the tube (FIG. 4).

[0081] One of the 2 bones is fixed at the bottom and the other at thetop of the tube. Then, the tube containing the bone plugs are filledwith sterile culture medium containing 10% FCS and put at 37° C.overnight in order to verify that no bacterial contamination comes out.Up to now, we never had any contamination following this method. Anotheralternative could be that the bones and thread would be rinsed with 100%ethanol, dried under sterile conditions and sterilized a second timewith ethylene oxide. They could be kept in sterile culture mediumcontaining 5-10% FCS at 4° C. until use.

[0082] Production of the Bioengineered ACL Substitutes in vitro

[0083] Two protocols have been developed to obtain similar products;graftable bioengineered ACL substitutes. The protocol involves theaddition of the living LF only at the end of the production steps,avoiding the use of cell-populated collagen gels during the.

[0084] A solution of DME 2.7X containing antibiotics is mixed with asecond solution containing heat inactivated (30 min at 56° C.) FCS,solubilized bovine Type I collagen and living LF (preferably frompassages 2 to 5; FIG. 10, step 3). The cells are added at a finalconcentration of 2.5×10⁵ cells per ml but lower or higher cellconcentrations could be used. The final concentration of bovine Type Icollagen varies between 1.0-2.0 mg/ml in the ACL substitutes but otherconcentrations could be used (e.g. preferably ranging from 0.5 to 5mg/ml). The next step is described on FIG. 10, step 5.

[0085] B) A solution of DME 2.7X containing antibiotics is mixed with asecond solution containing heat inactivated (30 min at 56° C.) FCS,solubilized bovine Type I collagen. The final concentration of bovineType I collagen varies between 1.0-2.0 mg/ml in the ACL substitutes butother concentrations could be used (e.g. preferably ranging from 0.5 to5 mg/ml). There is yet no cell added in the mixture at this stage.

[0086] The mixture is quickly poured in the sterile plastic tubecontaining the 2 bone anchors linked by the twisted surgical thread.

[0087] The ACL substitute is cultured in DME supplemented with 10% FCS,50 μg/ml ascorbic acid, 100 IU/ml penicillin G and 25 μg/ml gentamicin.It is maintained in a static vertical position during the first 24 hrsof culture mainly to allow proper collagen polymerization. The ACLsubstitute is then taken out of the tube after collagen polymerization(at pH 7.4). The collagen matrix is also contracted when the ACLsubstitute contained living LF according to procedure A (FIG. 5) but itis not contracted in the case of acellular substitutes prepared asdescribed in procedure B (FIG. 6).

[0088] Then, the ACL substitute is taken out of the tube and frozen at−80° C. in a sterile dish (FIGS. 10 and 11, step 5).

[0089] When frozen, the ACL substitute is lyophilized (FIG. 7 and FIGS.10 and 11, step 6).

[0090] The lyophilized ACL substitute is then transferred into a newsterile plastic tube and fixed as previously described to be used as asolid central core (FIGS. 10 and 11, step 7). Additional lyophilizedlayers can be added to produce larger and stronger ACL substitutes.

[0091] Another layer of hydrated collagen mixed with living LF is madeand added around the lyophilized collagen core, according to theprocedures described in section A. The bilayered ACL substitute can bekept in culture until grafted into the host. FIG. 8 shows a histologicalsection of the ACL substitute before implantation (transversal plan).The central lyophilized core is surrounded by a hydrated collagen layerseeded with LF of the eventual host, in that case, a goat.

[0092] A second layer of hydrated collagen is made as described insection B (no cell is included within the matrix) The acellular ACLsubstitute is a network of collagen fibers (FIG. 9A). After itspolymerization overnight, the acellular ACL substitute is put in culturemedium containing LF suspended in the medium (DME supplemented with 10%FCS, 50 μg/ml ascorbic acid, 100 IU/ml penicillin G and 25 μg/mlgentamicin; FIG. 11, step 8). Within 24 hrs, the cells attach andmigrate into the outer hydrated collagen layer (not lyophilized; FIG.9B). The cells contract the collagen matrix while colonizing it within48 hrs (FIG. 9C) The bilayered cell-populated ACL substitute can be keptin culture until grafted (FIG. 11, step 9). More hydrated matrix layerscan be added around the bACL.

[0093] Organization of Matricial Structure Induced in the ACL Substituteby Cyclic Traction

[0094] At least 10 replicates were conducted under similar conditions toevaluate the effects of cyclic traction on the evolution of our ACL. Thecycles were fixed at a frequency of 1 cycle/min. During the first 5days, the ACL were stretched to 1-mm stretch per cycle, always returningto their initial length (about 4 cm) to complete each cycle. Theamplitude was increased to 2 mm from days 5 to 10. Histologic studieswere performed after 10 days on ACL cultured under static horizontalconditions compared to ACL subjected to cyclic traction. For the firsttime, dense network of collagen fibers organized in wavy bundles isobserved in in a human bioengineered living tissue. Our data stronglyshow that living ACL cells seeded in ACL can respond to mechanicalstimuli in vitro. The crimps followed a wavy pattern, as it is seen innative ACL. Results were repeatedly similar from one experiment toanother.

[0095] Surgical Procedure for Implantation of the Bioengineered ACLSubstitutes in Human and Animals

[0096] Surgical procedures are performed by arthroscopy in human andunder general anesthesia in animals (intramuscular injection of ketamineand xylasine; 0.6 ml/kg body weight), maintained by inhalation of a 2:1mixture of oxygen and nitrous oxide with 0.1% halothane.

[0097] With use of Kirschner wires and a mini-driver, a tunnel (about 1cm diam., adapted to the knee of the host) will be created through themetaphyseal bone of the femur, distal to the epiphyseal scar andperpendicular to the long axis of the femur.

[0098] The bACL (about 1 cm length, adapted to the knee of the host) isplaced within the bone tunnel, with great care to ensure that the bACLfills the entire length of the hole.

[0099] The end of the prosthesis exiting the lateral end of the tunnelis inserted in a second tunnel performed in the lateral femoralperiosteum. A minimal static tension is applied on the bACL.

[0100] The bone anchors of the graft may be fixed with screws and/orcement (including biomedical epoxy).

[0101] The incision site is sprayed with a topical antibacterial agent.In the case of human, they receive a normal diet and movementrestrictions during the first month post-surgery. They start to putweight on the operated leg according to tolerance and receive anexercise program to maintain or increase muscular strength. Their kneesare monitored daily for a week to notice any abnormal inflammatorysigns. In the case of animals, they receive a diet of water and food adlibitum. Prophylactic tetracycline is added in water for 10 days. A castor a light orthosis presently used to limit human joint motionspostsurgery is used to prevent animal knee motion over 4-7 days afterbACL implantation. The animal's physical evaluation is done daily byveterinarians and their staff.

[0102] Such ligament substitute may be modified further or adapted forgene therapy by introducing genes into the cells. Also, the proceduremay be easily adapted to other applications, for example, to replace aligament at another anatomic site of the body (vertebral column, neck,etc).

EXAMPLE II Preparation of Connective Tissues

[0103] Material and Methods

[0104] Dermal Fibroblasts Isolation and Culture

[0105] The dermal fibroblasts (DF) isolated from the dermis of skinbiopsies, enzymatically (same procedure described in Example I) or byexplants, are cultured in DME supplemented with 10% fetal calf serum(FCS), 100 IU/ml penicillin G and 25 μg/ml gentamicin.

[0106] When DF primary cultures reach about 85% confluence, the cellsare detached from their culture flasks using 0.05% trypsin-0.01% EDTAsolution (pH 7.8), for about 10 min at 37° C. The DF suspensions arecentrifuged twice at 200×g for 10 min. The cell pellets are resuspendedin complete culture medium and the cells are counted. The cellularviability is determined using the trypan blue exclusion method.

[0107] Up until now, the DF were isolated and cultured from skinbiopsies of more than hundred patients and 10 animals (goats, dogs, andrabbits) with 100% success. The cells maintained their morphology formore than 7 passages in culture. For connective tissue substitutesproduction (e.g. ligaments), DF cultures from passages 2 to 5 are used.

[0108] Preparation of the Ligament Substitutes' Bone Anchors

[0109] Bone pieces are washed, cut and sterilized according to theprocedure described in Example I.

[0110] Holes are made in each bone anchor, as previously described. The2 sterile bone plugs readily linked by the twisted surgical thread aretransferred in a sterile plastic tube and kept in position by passing ahot metal pin through their transverse holes and across the tube (FIG.4).

[0111] Production of Bioengineered Ligament Substitutes in vitro

[0112] A first alternative: A solution of DME 2.7X containingantibiotics is mixed with a second solution containing heat inactivated(30 min at 56° C.) FCS, solubilized bovine Type I collagen and living DF(preferably from passages 2 to 5). The DF are added at a finalconcentration of 2.5×10⁵ cells per ml but lower or higher cellconcentrations could be used. The final concentration of bovine Type Icollagen varies between 1.0-2.0 mg/ml in the ligament substitutes butother concentrations could be used (e.g. preferably ranging from 0.5 to5 mg/ml). The next step is described in FIG. 11 step 5).

[0113] A second alternative: A solution of DME 2.7X containingantibiotics is mixed with a second solution containing heat inactivated(30 min at 56° C.) FCS, solubilized bovine Type I collagen. The finalconcentration of bovine Type I collagen varies between 1.0-2.0 mg/ml inthe ACL substitutes but other concentrations could be used (e.g.preferably ranging from 0.5 to 5 mg/ml). There is yet no cell added inthe mixture at this stage.

[0114] The mixture is quickly poured in the sterile plastic tubecontaining the 2 bone anchors linked by the twisted surgical thread.Collagen scaffolds are casted between two bone anchors described inexample I. The tissue constructs are put into a dessicator under minimalhorizontal tension, under normal atmospheric pressure or less (rangingfrom about 25 to 0 mm Hg). Tha appearance of the macroscopic aspect of abioengineered ACL ready for implantation can be seen in FIG. 12, as wellas immediately after implantation in situ (opened goat's knee joint)(FIG. 13). The scaffolds were completely dehydrated within about 2-3 hrs(FIG. 14). FIG. 15 shows a histological section of a collagen matrixdehydrated under these conditions.

[0115] The bioengineered scaffolds were rehydrated in fresh DMEM, takenout of the tube and then transferred into a new sterile plastic tube.Additional dehydrated layers can be added or another layer of hydratedcollagen can be added containing living DF or LF, to produce larger andstronger ligament substitutes.

[0116] An acellular bioengineered ligament has been grafted into agoat's knee joint. After five months, as shown in FIG. 16, the graftedligament is clearly colonized and innervated by the hosts' cells. Notethe presence of the host's cells which colonized the graftpost-implantation and the high density of collagen fibers, aligned inthe long axis of the regenerating anterior cruciate ligament in situ(longitudinal plan).

EXAMPLE III

[0117] Preparation of Periodontal Ligament Substitute

[0118] Material and Methods

[0119] Fibroblasts Isolation and Culture

[0120] Dermal fibroblasts (DF), ligament fibroblasts (LF), orfibroblasts from other sources (e.g. mucosa of the mouth) can beisolated and cultured in DME supplemented with 10% fetal calf serum(FCS), 100 IU/ml penicillin G and 25 μg/ml gentamicin.

[0121] When the cells primary cultures reach about 85% confluence, theyare detached from their culture flasks using 0.05% trypsin-0.01% EDTAsolution (pH 7.8), for about 10 min at 37° C. The cell suspensions arecentrifuged twice at 200×g for 10 min. The cell pellets are resuspendedin complete culture medium and the cells are counted. The cellularviability is determined using the trypan blue exclusion method.

[0122] Preparation of the Peridontal Ligament Substitutes' Tooth Anchors

[0123] Teeth pieces are washed and sterilized according to the proceduredescribed in Example I.

[0124] Holes are made in each tooth, as previously described. A steriletooth is linked to a bone anchor by a twisted surgical thread and bothare transferred in a sterile plastic tube and kept in position bypassing a hot metal pin through their transverse holes and across thetube.

[0125] Production of Bioengineered Peridontal Ligament Substitutes invitro

[0126] A solution of DME 2.7X containing antibiotics is mixed with asecond solution containing heat inactivated (30 min at 56° C.) FCS,solubilized bovine Type I collagen and living fibroblasts (preferablyfrom passages 2 to 5). The fibroblasts are added at a finalconcentration of 2.5×10⁵ cells per ml but lower or higher cellconcentrations could be used. The final concentration of bovine Type Icollagen varies between 1.0-2.0 mg/ml in the ligament substitutes butother concentrations could be used (e.g. preferably ranging from 0.5 to5 mg/ml).

[0127] According to a second possibility, a solution of DME 2.7Xcontaining antibiotics is mixed with a second solution containing heatinactivated (30 min at 56° C.) FCS, solubilized bovine Type I collagen.The final concentration of bovine Type I collagen varies between 1.0-2.0mg/ml in the ACL substitutes but other concentrations could be used(e.g. preferably ranging from 0.5 to 5 mg/ml). There is yet no celladded in the mixture at this stage.

[0128] The mixture is quickly poured in the sterile plastic tubecontaining the bone and the tooth anchors linked by the twisted surgicalthread. Collagen scaffolds are casted between two anchors. The tissueconstructs are lyophilized or put into a dessicator under minimalhorizontal tension, under normal atmospheric pressure or less (rangingfrom about 25 to 0 mm Hg). When totally dehydrated, the scaffolds arerehydrated in fresh DMEM, taken out of the tube and then transferredinto a new sterile plastic tube. Another layer of hydrated collagen canbe added containing living fibroblasts, to produce larger and strongerligament substitutes. The periodontal ligament substitute can beimplanted in the gum (FIG. 17).

[0129] While the invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

What is claimed is:
 1. An implant for connective tissue substitution inan animal, said implant comprising a pair of bone anchors joined attheir proximal ends by at least one support filament, said supportfilament being coated by at least one matrix layer of thicknesssufficient to allow for colonization by a cell.
 2. The implant accordingto claim 1, wherein said matrix layer is colonized by a cell.
 3. Theimplant according to claim 1, wherein said connective tissuesubstitution is partial or complete substitution of a connective tissue.4. The implant according to claim 1, wherein said connective tissue isselected from the group consisting of a tendon, a cartilage, a disk, ameniscus, a muscle, a tooth, a hair, a joint, and a ligament, or acombination thereof.
 5. The implant according to claim 1, wherein saidanimal is a human.
 6. The implant according to claim 1, wherein saidanimal is a non-human mammal.
 7. The implant according to claim 1,wherein said bone anchor is selected from the group consisting of a boneportion and a piece composed of at least one of natural or syntheticbiocompatible porous material.
 8. The implant according to claim 1,wherein said matrix layer is a collagen gel layer.
 9. The implantaccording to claim 1, wherein said matrix layer is selected from thegroup consisting of chitosan, glycosaminoglycan, chitin, ubiquitin,elastin, polyethylen glycol, polyethylen oxide, vimentin, fibronectin,and a protein promoting collagen alignment or assembly, or derivativesor a combination thereof.
 10. The implant according to claim 1, whereinsaid support filament is selected from the group consisting of at leastone of a resorbable thread, a natural fiber, and a filament composed ofa protein, a lipid, a biocompatible molecule, and a synthetic component.11. The implant according to claim 1, wherein said matrix layer furthercomprises a cell.
 12. The implant according to claim 1, wherein cell isan autologous cell.
 13. The implant according to claim 1, wherein saidcell is a heterologous cell.
 14. The implant according to claim 1,wherein said cell is selected from the group consisting of a fibroblast,a myoblast, an osteoblast, a mesenchymal cell, an endothelial cell, animmune cell, and a chondrocyte cell, or a combination thereof.
 15. Theimplant according to claim 1, wherein said matrix layer furthercomprises a pharmaceutically effective amount of biologically activemolecule selected from the group consisting of a drug, a growth factor,a cytokine, an antibiotic, and a hormone, or a combination thereof. 16.The implant according to claim 1, wherein at least one of said supportfilament or matrix layer is dehydrated or lyophilized prior toimplantation.
 17. The implant according to claim 1, wherein said matrixlayer further comprises at least one inner layer of gel and/or filamentcoated by at least one supplementary matrix coating layer.
 18. Theimplant according to claim 17, wherein said inner layer is dehydratedand said filament is hydrated and lyophilized prior to be incorporatedto said matrix layer.
 19. The implant according to claim 17, whereinsaid implant is dehydrated or lyophilized prior coating with saidsupplementary matrix coating layer.
 20. The implant according to claim19, wherein said supplementary matrix coating layer is furtherdehydrated or lyophilized before being coated by another supplementarymatrix coating layer.
 21. The implant according to claim 17, whereinsaid matrix coating layer further comprises a cell.
 22. The implantaccording to claim 17, wherein said cell is an autologous cell.
 23. Theimplant according to claim 21, wherein said cell is a heterologous cell.24. The implant according to claim 21, wherein said cell is selectedfrom the group consisting of a fibroblast, a myoblast, an osteoblast, amesenchymal cell, an endothelial cell, an immune cell, and achondrocyte, or a combination thereof.
 25. A method of preparing animplant for connective tissue substitution in an animal, said methodcomprising the steps of: a) providing a set of bone anchors by joining apair of bone plugs at their proximal ends by at least one supportfilament; and b) incubating at least one time said set of bone anchorsof step a) in a solution containing matrix forming molecules for aperiod time sufficient for the formation of at least one matrix layeraround said support filament, wherein said matrix layer has a thicknesssufficient to allow for colonization by cells, and wherein saidincubation is performed under conditions in which are induced waves,vibrations, cyclic tractions, and/or static tractions of said implant.26. The method according to claim 25, wherein said matrix is furthercolonized by a cell.
 27. The method according to claim 25, wherein saidimplant is dehydrated, lyophilized and/or chemically treated prior toimplantation.
 28. The method according to claim 25, wherein saidconnective tissue is selected from the group consisting of a tendon, acartilage, a disk, a meniscus, a muscle, a tooth, a hair, a joint, and aligament, or a combination thereof.
 29. The method according to claim25, wherein said animal is a human.
 30. The method according to claim25, wherein said animal is a non-human mammal.
 31. The method accordingto claim 25, wherein said bone anchor is selected from the groupconsisting of a bone portion, and a piece composed of natural and/orsynthetic biocompatible porous material.
 32. The method according toclaim 25, wherein said matrix layer is a collagen gel layer.
 33. Themethod according to claim 25, wherein said matrix layer is composed ofcompound selected from the group consisting of chitosan,glycosaminoglycan, chitin, ubiquitin, elastin, polyethylen glycol,polyethylen oxide, vimentin, and fibronectin, or derivatives orcombinations thereof.
 34. The method according to claim 25, wherein saidfilament is selected from the group consisting of a resorbable thread, anatural fiber, and a filament composed of at least one of protein,lipid, biocompatible molecule or synthetic component.
 35. The methodaccording to claim 25, wherein said matrix layer further comprises cell.36. The method according to claim 25 or 26, wherein said cell is aheterologous cell.
 37. The method according to claim 25 or 26, whereinsaid cell is selected from the group consisting of a fibroblast, amyoblast, an osteoblast, a mesenchymal cell, an endothelial cell, animmune cell, a chondrocyte, and a combination thereof.
 38. The methodaccording to claim 25, wherein said matrix further comprises apharmaceutically effective amount of biologically active moleculeselected from the group consisting of a drug, a growth factor, acytokine, an antibiotic, a hormones, and a combination thereof.
 39. Themethod according to claim 25, wherein said inner matrix layer is coatedby at least one supplementary matrix coating layer.
 40. The methodaccording to claim 39, wherein at least one of said inner matrix layeror filament is dehydrated or lyophilized prior coating by saidsupplementary matrix coating layer.
 41. The method according to claim39, wherein said supplementary matrix coating layer is dehydrated orlyophilized before being coated by another supplementary matrix coatinglayer.
 42. The method according to claim 39 or 41, wherein saidsupplementary matrix coating layer or another supplementary matrixcoating layer further comprises a cell.
 43. The method according toclaim 25, wherein said cell is an autologous cell.
 44. The methodaccording to claim 25, wherein said cell is a heterologous cell.